ABSTRACT The long-term goal of this proposal is to study mechanism(s) of pathogenicity of Cryptococcus neoformans (Cn) focusing on how Cn glycolipids regulate fungal virulence and the host immune response. Cn is a fungal pathogen that, upon entering the lung and disseminating through the bloodstream, causes a life-threatening meningo-encephalitis in susceptible patients, leading to high morbidity and mortality. Current therapies for this disease can have intolerable and dose-limiting side effects.1 Thus, new treatment strategies are warranted to better control the high mortality associated with cryptococcosis. Although vaccines have been hailed as one of the greatest achievements in public health during the past century, the development of safe and efficacious vaccines against cryptococcosis, and fungal infections in general, has been a major hurdle mainly due to the lack of knowledge about the mechanisms that underpin protective immunity. Additionally, fungal vaccines need to be effective in conditions of immunodeficiency, such as CD4+ T cell lymphopenia in AIDS patients, because immunodeficient patients are the most susceptible to cryptococcosis and other invasive fungal infections. In previous studies, we deleted the sterol-glucosidase 1 (Sgl1) gene and the resulting mutant (?sgl1) is highly enriched in sterol glucosides (SGs) (Fig. 3 and 4), which are otherwise not detectable in wild-type Cn cells. SGs are glycolipids present in a variety of fungi and other microorganisms and are prone to stimulate host immunity.2-4 We found that the Cn ?sgl1 mutant is not pathogenic in a mouse model (Fig. 8A) and, upon intranasal administration, the mutant cells are rapidly killed in the lung environment (Fig. 8B). Very interestingly, mice receiving Cn ?sgl1 mutant are protected when challenged with virulent Cn H99 or C. gattii 265 strains (Fig. 10), and this protection persists even when mice are CD4+ T cell depleted (Fig. 12). Based on these observations, we hypothesize that Cn Sgl1 is a major regulator of fungal virulence by modulating the level of SGs, which in turn stimulate the host immune response against cryptococcosis. To test this hypothesis we propose the following aims: Aim 1. To establish Sgl1 as a key regulator of SGs in fungi. In this aim 1 we will: 1A) study the biochemical properties of Sgl1; and 1B) study the role of Sgl1 in regulating SGs and virulence. Aim 2. To determine the host immune mechanisms against ? sgl1. In this aim we will: 2A) examine the immune response leading to ? sgl1 clearance; 2B) examine the immune response protecting the ? sgl1-immunized mice to secondary infection; and 2C) use depletion approaches to examine the cellular requirement for initial clearance (2A) and protection from secondary challenge infection (2B). This proposal will provide the first insights in the role and mechanisms by which fungal SGs regulate the patho-biology of an important human fungal pathogen, namely Cn. They will also potentially lead to the development of novel antifungal strategies to prevent or/and treat invasive fungal infections in immunocompetent and immunocompromised hosts.